Coaxial Flow Device for Nanoparticle Preparation and Manufacturing Equipment Including Such Device

The present invention relates to equipment and processes for the manufacturing of nanoparticles.

More specifically the invention relates to a coaxial flow device capable of creating comparable microenvironments at various operation scales through the continuous introduction and mixing of nanoparticle precursor solutions for the manufacturing of a dispersion comprising nanoparticles. The nanoparticles may optionally include an encapsulated payload.

In the pharmaceutical field, an increasing number of promising gene therapies and vaccines are based on RNA and DNA polymers. A critical issue associated with the implementation of such RNA- or DNA-based gene therapies or vaccines is delivery. Naked RNA or DNA molecules are rapidly degraded in biological fluids, do not accumulate in tissues following systemic administration, and cannot penetrate target cells, even if they get to the target tissues. Further, the immune system is designed to recognize and destroy vectors containing genetic information.

It has therefore been proposed to administer RNA or DNA molecules encapsulated in lipid nanoparticles (LNPs) such that the RNA or DNA molecules can be delivered to the target cells without degradation.

In the case of an RNA vaccine, LNPs aid delivery of RNA to cells and thereby promote an immunological response. The formation of the LNPs and the encapsulation of the RNA is critical to the efficacy of the vaccine and the manufacturing operations bringing the RNA and the lipid material together must be done in appropriate conditions to enable proper encapsulation.

Conventional in-line mixing devices, commercially available for the mixing of two pressurized or controlled fluid streams in a production line equipment in the pharmaceutical field, include so-called “tee mixer-type connectors”. The term “tee mixer-type connector” refers to a hydraulic connector designed to connect two tubes, possibly with different diameters, to combine fluid flows from these tubes and change their direction. It includes two opposing inlets oriented in substantially parallel directions and an outlet oriented in a substantially perpendicular direction. The inlets receive the flows from the two distinct tubes and these flows combine in the outlet. The two fluid flows from the connecting tubes may have different velocities. The term “tee mixer-type connector” encompasses such connectors forming a T shape (“T-mixer” or “T-connector”) and those forming a Y shape (“Y-mixer” or “Y-connector”).

Such mixing devices, while convenient for laboratory equipment or relatively small-scale production lines, cannot be adapted to high throughput and large scale production.

There is a requirement for production line equipment and more specifically for a mixing device to be able to combine two fluid streams, such as an RNA aqueous stream with one or more lipid organic stream(s), in a continuous and reproducible way, at various production scales. This is a particularly essential requirement for the production of vaccines in the context of a pandemic, wherein the vaccines need to be rapidly made available to the greatest number.

According to a first aspect of the present invention, it is provided a coaxial flow device capable of creating comparable microenvironments at various operation scales through the continuous introduction and mixing of nanoparticle precursor solutions for the manufacturing of a dispersion comprising nanoparticles, the device including

According to optional features, which may be considered separately or in every technically meaningful combination:

In a further aspect of the invention, it is provided an equipment for the manufacturing of a dispersion comprising nanoparticles including an encapsulated payload, comprising

The following definitions will be used in the present description and claims:

The invention will now be further illustrated by the following preferred embodiments, corresponding to a large scale coaxial flow mixing device and a manufacturing equipment including such a coaxial flow device that can be used for the commercial manufacturing of a formulation comprising lipid nanoparticles, optionally including a payload.

In particular, but not necessarily, the payload may be a polynucleotide. Also, the payload may include entities of one or more types.

In a particular application of the invention, the coaxial flow device may be used for the manufacturing of a formulation used in an mRNA vaccine.

Suitable lipids and polynucleotides for use with the coaxial flow device and manufacturing equipment of the invention are exemplified below.

The lipid component of a LNP may include, for example, a cationic lipid, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC), a PEG lipid, and a structural lipid. The elements of the lipid component may be provided in specific fractions.

In some examples, the LNP further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof. Suitable phospholipids, PEG lipids, and structural lipids are further disclosed herein.

In some examples, the lipid component of a LNP includes a cationic lipid, a phospholipid, a polymer-conjugated lipid (e.g. polyethylene glycol (PEG)) and a structural lipid. In certain examples, the lipid component of the lipid nanoparticle includes about 30 mol % to about 60 mol % cationic lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In some examples, the lipid component of the lipid nanoparticle includes about 35 mol % to about 55 mol % compound of cationic lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid. In a particular example, the lipid component includes about 50 mol % said cationic lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular example, the lipid component includes about 40 mol % said cationic lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In some examples, the phospholipid may be DOPE or DSPC. In other examples, the PEG lipid may be PEG-DMG and/or the structural lipid may be cholesterol.

The amount of a therapeutic and/or prophylactic in a LNP may depend on the size, composition, desired target and/or application, or other properties of the lipid nanoparticle as well as on the properties of the therapeutic and/or prophylactic. For example, the amount of an RNA useful in a LNP may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and/or prophylactic and other elements (e.g., lipids) in a LNP may also vary. In some examples, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic in a LNP may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1,8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic may be from about 10:1 to about 40:1. In certain examples, the wt/wt ratio is about 20:1. The amount of a therapeutic and/or prophylactic in a LNP may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

In some examples, the ionizable lipid is a compound of Formula (IL-I):

or their N-oxides, or salts or isomers thereof,

In preferred embodiments, the cationic lipid is a compound having the following structure (IE):

In some embodiments, the compound includes the following structure:

wherein Ris, at each occurrence, H; n is an integer ranging from 2 to 12; and y and z are each independently integers ranging from 6 to 9. In some embodiments, n is 3, 4, 5 or 6. 4. In some embodiments, y and z are each 6. In some embodiments, y and z are each 9. In some embodiments, Rand Reach, independently has the following structure wherein: Rand Rare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R, Rand a are each selected such that Rand Reach independently comprise from 6 to 20 carbon atoms. In some embodiments, a is an integer from 8 to 12. In some embodiments, at least one occurrence of Ris H. In some embodiments, Ris H at each occurrence. In some embodiments, at least one occurrence of Ris C1-C8 alkyl. In some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl. In some embodiments, Ris OH. In some embodiments, Ris CN. In some embodiments, Ris —C(═O)OR4, —OC(═O)Ror NHC(═O)R. In some embodiments, Ris methyl or ethyl. In some embodiments, the compound has the following structure:

Additional exemplary ionizable lipids include:

The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some examples, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some examples, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some examples, the PEG-modified lipids are a modified form of PEG DMG. In some examples, the PEG-modified lipid is PEG lipid with the formula (IV):

wherein Rand Rare each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In some embodiments, the polymer-conjugated lipid is a polyoxazoline (POZ) lipid comprising the formula (IV):

POZ is known in the art and is described in WO/2020/264505, PCT/US2020/040140, filed on Jun. 29, 2020.

In some embodiments, the PEGylated lipid has the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Rand Rare each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60; provided that Rand Rare not both n-octadecyl when z is 42. In some embodiments of the PEGylated lipid, Rand Rare each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, of the pegylated lipid z is about 45.

In some embodiments, the PEGylated lipid has one of the following structures:

wherein n has a mean value ranging from 40 to 50. In a preferred embodiment, the composition comprises the ALC-315 cationic lipid described above and a PEGylated lipid having one of the following structures:

In some embodiments of the PEGylated lipid described above, Rand Rare each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments of the PEGylated lipid described above, Rand Rare each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In some embodiments of the PEGylated lipid described above, Rand Rare each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. Further exemplary lipids and related formulations thereof are disclosed for example, in U.S. Pat. No. 9,737,619, filed Feb. 14, 2017, U.S. Pat. No. 10,166,298, filed Oct. 28, 2016, and International Patent Application No. PCT/US2017/058619, filed Oct. 26, 2017, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the ionizable lipid is a compound of Formula (IL-I):

In some examples, a LNP includes one or more polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide,” in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. In some examples, a therapeutic and/or prophylactic is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), self-amplifying RNA (saRNA), and mixtures thereof. In certain examples, the RNA is an mRNA.

In certain examples, a therapeutic and/or prophylactic is an mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some examples, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

In other examples, a therapeutic and/or prophylactic is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a LNP including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some examples, the siRNA may be an immunomodulatory siRNA.